The Notch pathway functions as a cell signaling mechanism that regulates tissue patterning and morphogenesis in developing vertebrates and invertebrates. Notch activity is a central element of neurogenesis and neurite outgrowth, and has been implicated in the etiology of Alzheimers Disease. The overall objective of my research is to understand the extracellular molecular events that regulate Notch receptor signaling. This proposal will examine the role of the ADAM (a disintigrin and metalloprotease) family of metalloproteases in modulating Notch activity. Our previous studies have shown that several cleavage events, which occur extracellularly in Notch and Delta, are pivotal to activation of the pathway. Two of these cleavages have been ascribed to the ADAM metalloproteases known as Kuzbaman (Kuz) and TNF-a converting enzyme (TACE), respectively. We have recently identified novel cleavages in Notch and Delta for which an enzyme has yet to be defined. The proposed studies are based in a Drosophila model to perform a comprehensive analysis of the role of the ADAM family of metalloproteases in regulating Notch signaling activity in neurogenesis. We will first characterize the expression profile of the ADAMs, Notch, Delta and the downstream Enhancer-of-Split genes in the CNS as an essential first step in establishing the molecular relationship of these proteases with the Notch pathway. An examination of the discrete biochemical activity of the ADAMs toward Notch and Delta will follow utilizing a defined expression system that we have successfully employed to identify the enzyme-substrate relationship between Kuz and Delta. We will translate this expertise to evaluate processing events in the ADAMs themselves, which we anticipate will advance our understanding of the presentation of ADAM metalloprotease activity in the cellular environment. These analyses will be coupled with an evaluation of ADAM activity toward Notch signaling in two distinct developmental contexts in neurogenesis in vivo. Overall, these experiments represent a multi-faceted approach to characterizing an emerging concept in signal transduction, namely, the regulation of signaling activity by proteolysis at the cell surface.